The integrase genes of leukemogenic MCF13 and non-leukemogenic MCFM111A murine retroviruses were compared in their structure and biological activities. Nucleotide sequence analysis indicated that the two integrase genes were distinct: there were 21 substituted amino acids between MCF13 and MCFM111A, of which 4 affected the hydropathicity profile; in addition, 4 amino acids were present in MCF13 murine leukemia virus (MuLV) which were absent in MCFM111A MuLV. To determine whether the structural differences seen between the integrase genes of leukemogenic versus non-leukemogenic murine viruses affected integrase function, we analyzed the integrase protein activities using an in vitro biological system. This assay measured the incorporation of linear viral DNAs, present in cytoplasmic extracts from infected cells, into phi-X174 target DNA. The results indicated that integration by leukemogenic MCF13 was two-fold greater than by non-leukemogenic MCFM111A.